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N Lehman

Publications and source records attributed to N Lehman.

At least 19 recordsLinked to original sources
PubMed2006

The elucidation of novel SH2 binding sites on PLD2.

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y(169) and Y(179) in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y(169) and Y(179) are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y(169) (PLD2 Y169F) or Y(179) (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y(169) serves to modulate the activity of the enzyme, whereas Y(179) regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y(169) and Y(179) are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42(Erk) phosphorylation and enhanced DNA synthesis. Since Y(169) remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y(169) is kept under negative regulation by Y(179). When this is released, Y(169) mediates cellular proliferation through the Ras/MAPK pathway.

Amino Acid Sequence↗
PubMed2005

Conservation of MHC class II DOA sequences among carnivores.

We obtained the nucleotide sequence for most of the major histocompatibility complex (MHC) class II DOA locus for Weddell, leopard, northern elephant, and southern elephant seals and from the coyote and compared them to all known DOA data available to date. We found generally low levels of interspecific polymorphisms, providing further support for stabilizing selection acting on the DOA locus. This suggests that DO gene products play a substantial functional role in the regulation of antigen presentation. A seven-amino-acid motif of VWRLPEF was found to be conserved across all DOA sequences and may be a DO-specific recognition element.

Alleles↗
PubMed2002

Major histocompatibility complex class II DOA sequences from three Antarctic seal species verify stabilizing selection on the DO locus.

To provide additional support for the sequence conservation and hence the regulatory role of the MHC class II DOA locus, we obtained the nucleotide sequences of exon 2 and exon 3, along with the intervening intron, of the Ross seal, and sequences from the exon 2 region from the Weddell and leopard seals. These are the first reports of the sequences of this locus from a carnivore species. The results demonstrate strong conservation among mammals for the exon sequence and produce a gene genealogy that is consistent in topology with a species tree.

Amino Acid Sequence↗
PubMed2001

Molecular evolution: Please release me, genetic code.

The genetic code is no longer universal, even in non-mitochondrial genomes. Recent studies have implicated the eukaryotic release factor eRF1 in mediating coding changes that are not as inconceivable as once thought. Specific residues in eRF1 proteins can be correlated with specific code changes in a wide variety of taxa.

Animals↗
PubMed2000

An empirical genetic assessment of the severity of the northern elephant seal population bottleneck.

A bottleneck in population size of a species is often correlated with a sharp reduction in genetic variation. The northern elephant seal (Mirounga angustirostris) has undergone at least one extreme bottleneck, having rebounded from 20-100 individuals a century ago to over 175,000 individuals today. The relative lack of molecular-genetic variation in contemporary populations has been documented, but the extent of variation before the late 19th century remains unknown. We have determined the nucleotide sequence of a 179 base-pair segment of the mitochondrial DNA (mtDNA) control region from seals that lived before, during and after a bottleneck low in 1892. A 'primerless' PCR was used to improve the recovery of information from older samples. Only two mtDNA genotypes were present in all 150+ seals from the 1892 bottleneck on, but we discovered four genotypes in five pre-bottleneck seals. This suggests a much greater amount of mtDNA genotypic variation before this bottleneck, and that the persistence of two genotypes today is a consequence of random lineage sampling. We cannot correlate the loss of mtDNA genotypes with a lowered mean fitness of individuals in the species today. However, we show that the species historically possessed additional genotypes to those present now, and that sampling of ancient DNA could elucidate the genetic consequences of severe reductions in population size.

Animals↗
PubMed2000

The genotypic landscape during in vitro evolution of a catalytic RNA: implications for phenotypic buffering.

The Tetrahymena group I ribozyme catalyzes the cleavage of a phosphodiester linkage in specific sequences of RNA. This phenotype can be used in an in vitro selection-amplification process to evolve variants that are capable of RNA catalysis in the presence of Ca(2+) as the sole available cation. With sufficient genotypic characterization of the population as it evolves, we have a rare opportunity of observing how the information stored in an evolving population responds to selective pressures, such as the requisite of catalyzing RNA cleavage in the absence of Mg(2+) or Mn(2+). In the present work, we examine the population dynamics of this system using sequence information from previous experimental work. We focus on two issues: How does the information content of the population evolve? and Is the system evolving as an adaptive walk on a rugged landscape? To investigate these questions, information theoretical parameters are examined. The evolution of the population is visualized by mapping the genotypic frequency distribution onto a two-dimensional projection of sequence space. The projection was generated using Hamming distances from the wild-type, starting sequence and a catalytically successful, evolved sequence. The evolution of the information content of the system was measured by calculating the grammar complexity of the observed sequences, which showed a very slight increase over 12 generations. This result is consistent with the system performing a search for a local optimum. The dynamics of the population in this sequence space is consistent with an adaptive walk on an uncorrelated, or "rugged," genotypic landscape, despite the observation that the phenotypic progress of the population appears smooth. The relative insensitivity of the phenotypic landscape to the variegation of the genotypic landscape suggests that the former is buffered against variation in the latter through various epigenetic-like mechanisms.

Evolution, Molecular↗
PubMed2000

Multi-locus genetic evidence for rapid ecologically based speciation in Daphnia.

The process of speciation involves the divergence of two or more subpopulations of a parent species into independent evolutionary trajectories. To study this process in natural populations requires a detailed knowledge of the genetic and ecological characteristics of the parent species and an understanding of how its populations can lose evolutionary cohesion. The cosmopolitan and speciose genus Daphnia provides many of these features by existing in multiple freshwater habitat types, particularly permanent lakes and temporary ponds, each of which presents distinct ecological challenges. We assayed the genetic composition of 20 temporary pond populations of members of the Daphnia pulex species complex in north-western Oregon and compared them to published data on related lake and pond populations. We collected molecular genetic data from 13 allozyme loci, from six microsatellite loci, and from the control region of the mitochondrial DNA. By assaying over 400 individual Daphnia for these data, we were able to compile composite genotypes not only of individual Daphnia but of each pond population as a whole. In these ponds, we discovered two distinct genotypic constellations, one which bears resemblance to the lake-dwelling taxon D. pulicaria, and one which bears resemblance to the pond-dwelling taxon, D. pulex. Using published genetic data from these and other species as a frame of reference, we characterized 13 of these ponds as being 'pond-like', three as being 'lake-like', and four as being 'mixed'. Unlike studies performed elsewhere, however, these ponds do not exhibit high probabilities of interspecific hybridization. Over 95% of all individuals have either a lake-like or a pond-like genotype at all three genetic systems, suggesting the two forms do not represent hybridized vs. nonhybridized genotypes. Because both types can be found in the same ponds at the same time in gametic disequilibrium, we also discount the possibility that they are two extremes of a single species that is highly genetically subdivided. With these genetic data, and with supporting life-history and ecological data previously gathered on these pond populations, we conclude that the most likely description of this system is of a taxon caught in the act of speciating, with new pond-adapted populations periodically stemming from lake-adapted sources during river flooding events.

Alleles↗
PubMed1999

Non-unity molecular heritability demonstrated by continuous evolution in vitro.

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

Base Sequence↗
PubMed1998

Conservation biology: genes are not enough.

A study of correlated genotypic and phenotypic changes over a 2400-year period in a cave population of pocket gophers bolsters the idea that small, isolated populations can not only persist in a fluctuating environment, but may be able to adapt without genetic input from elsewhere.

Animals↗
PubMed1998

A randomized nearest-neighbor approach for assessment of character displacement: the vulture guild as a model

Character displacement has been analysed in the past but not with an approach that considers nearest-neighbor distances between members of a guild. Vultures provide a model system in which to test a new analytical approach presented here to assess character displacement. Vultures are represented by two geographically isolated and taxonomically distinct groups: the Old World accipitrids and New World vultures. These two groups provide an excellent case of convergent evolution in which functional similarities can be compared among obligate carrion-feeding birds. The vulture guild was analysed from several geographic regions where species occur in a high diversity: East Africa, South Africa, the Indian subcontinent, Amazonia, and the pleistocene deposits from Rancho La Brea, California. A three-dimensional morphospace was constructed derived from features of the skull, beak, and mandible to assess feeding capabilities. Species packing and their distribution within the morphospace were compared using a nearest-neighbor approach through Monte Carlo simulations. Vultures seem to exhibit a similar array of ecomorphological types wherever they occur in a high diversity, even though there are phylogenetic differences among some regions. Phylogenetic effects appear to have little influence on the distribution of functional types in each region and evidence for character displacement was found only at Rancho La Brea where both Old and New World vultures were present.Copyright 1998 Academic Press Limited Copyright 1998 Academic Press Limited

Journal Article↗
PubMed1995

A hierarchical molecular phylogeny within the genus Daphnia.

The nucleotide sequences of two mitochondrial DNA regions were used to determine phylogenetic relationships in the genus Daphnia (water fleas), a group for which systematics are historically unstable. A portion of the small ribosomal RNA was used to reconstruct higher-level relationships among species, while a portion of the more rapidly evolving control region was used to reconstruct lower-level relationships among populations. Two unexpected results were obtained. First, the subgeneric status of Ctenodaphnia falls into uncertainty on the basis of the failure of the 12S rRNA sequences to support these species as comprising an outgroup to the remainder of Daphnia species. Second, the high similarity of 12S rRNA sequences of Daphnia pulex and Daphnia pulicaria samples, coupled with the dual paraphyly of these samples as reconstructed from control-region analysis, suggests that they are two clonotype constellations within the same species complex. A combination of a variety of ecological influences has apparently resulted in the evolution of sets of Daphnia genotypes that are genetically cohesive despite their phenotypic divergence.

Alleles↗
PubMed1993

Evolution in vitro of an RNA enzyme with altered metal dependence.

The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

Animals↗
PubMed1993

Evolution in vitro: analysis of a lineage of ribozymes.

BACKGROUND: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. RESULTS: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. CONCLUSIONS: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

Animals↗
PubMed1991

Analysis of coyote mitochondrial DNA genotype frequencies: estimation of the effective number of alleles.

A restriction-site survey of 327 coyotes (Canis latrans) from most parts of their North American range reveals 32 mitochondrial DNA (mtDNA) genotypes. The genotypes are not strongly partitioned in space, suggesting that there is high gene flow among coyote subpopulations. Consequently, each new geographic location added to the study has a decreasing probability of containing a mtDNA genotype that had not been previously discovered. This being the case, by using Monte Carlo sampling experiments, we can estimate the total number of genotypes that would be found if all possible localities were surveyed. This estimate of total genotypic variability agrees qualitatively with estimates based on theoretical considerations of the expected number of alleles in a stable population. We also predict effective population sizes from genotype data. The accuracy of these estimates is thought to be dependent on the fact that coyotes are not highly genetically structured, a situation which may apply to highly mobile species.

Alleles↗
PubMed1990

Genetic fingerprinting reflects population differentiation in the California Channel Island fox.

Restriction fragment profiles generated by hybridization of hypervariable minisatellite DNA probes have been used for paternity analysis but not for comparisons at the level of populations, because the profiles are thought to evolve too rapidly to be informative over large time intervals. But in small isolated populations, the fixation of restriction-fragment polymorphisms can outpace the generation of fragment-length variability through recombination. Here we report on an analysis of DNA fingerprints of the California Channel Island fox (Urocyon littoralis). These foxes comprise an island dwarf species found only on six of the Channel Islands off the coast of southern California. Variability of restriction-fragment profiles within fox populations, as indicated by the average percentage difference (APD), varied widely among the islands, from 0.0% (no variation) to 25.3%. The APDs between populations were considerably greater (43.8% to 84.4%). In addition, foxes on each island can be distinguished by the presence of diagnostic restriction fragments. Maximum parsimony and phenetic trees relating foxes from different islands are consistent with the archaeozoological and geological record. Therefore, in small populations of genetically isolated mammals, differences among hypervariable restriction-fragment profiles can be used to estimate relative genetic variability and to reconstruct the evolutionary relationships of natural populations.

Animals↗
PubMed1990

Large sequence divergence among mitochondrial DNA genotypes within populations of eastern African black-backed jackals.

In discussions about the relative rate of molecular evolution, intraspecific variability in rate is rarely considered. An underlying assumption is that intraspecific sequence differences are small, and thus variations in rate would be difficult to detect or would not affect comparisons among distantly related taxa. However, several studies on mammalian mitochondrial DNA (mtDNA) have revealed considerable intraspecific sequence divergence. In this report, we test for differences in the rate of intraspecific evolution by comparing mtDNA sequences, as inferred from restriction site polymorphisms and direct sequencing, between mtDNA genotypes of the eastern African black-backed jackal, Canis mesomelas elongae, and those of two other sympatric jackal species. Our results are unusual for several reasons. First, mtDNA sequence divergence within several contiguous black-backed jackal populations is large (8.0%). Previous intraspecific studies of terrestrial mammals have generally found values of less than 5% within a single population, with larger divergence values most often occurring among mtDNA genotypes from geographically distant or isolated localities. Second, only 4 mtDNA genotypes were present in our sample of 64 jackals. The large sequence divergence observed among these mtDNA genotypes suggests there should be many more genotypes of intermediate sequence divergence if they had evolved in sympatry. Finally, estimates of the rate of mtDNA sequence evolution differ by approximately 2- to 4-fold among black-backed jackal mtDNA genotypes, thus indicating a substantial heterogeneity in the rate of sequence evolution. The results are difficult to reconcile with ideas of a constant molecular clock based on random fixation of selectively neutral or nearly neutral mtDNA sequence mutations.

Africa, Eastern↗
PubMed1988

Genetic code development by stop codon takeover.

A novel theoretical consideration of the origin and evolution of the genetic code is presented. Code development is viewed from the perspective of simultaneously evolving codons, anticodons and amino acids. Early code structure was determined primarily by thermodynamic stability considerations, requiring simplicity in primordial codes. More advanced coding stages could arise as biological systems became more complex and precise in their replication. To be consistent with these ideas, a model is described in which codons become permanently associated with amino acids only when a codon-anticodon pairing is strong enough to permit rapid translation. Hence all codons are essentially chain-termination or "stop" codons until tRNA adaptors evolve having the ability to bind tightly to them. This view, which draws support from several lines of evidence, differs from the prevalent thinking on code evolution which holds that codons specifying newer amino acids were derived from codons encoding older amino acids.

Amino Acids↗